1. Field Of the Invention
This invention in general relates to optical scanning systems and, in particular, to an apparatus and method for aligning and positioning microarray samples in a microarray scanning system.
2. Description Of The Prior Art
Microarray samples are being increasingly used for the performance of large numbers of closely-related chemical tests. Reference or xe2x80x98targetxe2x80x99 DNA (or RNA) is deposited as an array of target spots, or samples, onto a glass substratexe2x80x94typically a one-by three-inch glass microscope slidexe2x80x94where the genetic material chemically binds to the substrate surface. Each target spot of genetic material constitutes the locus of a separate experiment. xe2x80x98Probexe2x80x99 DNA (or RNA) containing a fluorophore material is then added to some or all of the target spots and is allowed to hybridize with the target material. Excess probe material that does not hybridize with and bind to target material is removed from the microarray sample surface in a subsequent washing process.
The experiments measure the binding affinities between probe DNA and target DNA, for example, to determine the similarity in their molecular structures; complementary molecules have a much greater probability of binding than unrelated molecules. The fluorophore present in the probe material emits a range of radiation energy centered about a wavelength xcexemission in response to illumination by an incident radiation source of a particular, shorter wavelength xcexexcitation. The brightness of emitted radiation, as measured by the detection system of the microarray scanning system, is a function of the fluorophore concentration present in the illuminated sample. Because the fluorophore concentration is a function of the binding affinity or likeness of the probe molecule to the target molecule, the brightness of a hybridized spot is an indication of the degree of similarity between the probe genetic material and the target genetic material present. A typical microarray sample may allow up to tens of thousands of experiments to be performed simultaneously on the genetic material, thus producing a detailed characterization of a particular gene under investigation.
In a microarray scanning system, the area of interest is usually divided into an array of discrete elements referred to as xe2x80x98pixels.xe2x80x99 Each pixel is illuminated independently as it is being addressed by the scanning system. The excitation radiation source is typically a single-wavelength laser device focused down to form an excitation radiation spot of the desired size. Emission radiation is emitted by the illuminated fluorophore in an outward, spherical beam. A portion of this emission beam is collected by an optical system and transmitted to a detector. In addition to the emitted radiation, some of the incident excitation radiation scattered from the surface of the sample is also collected by the optical system. To minimize the amount of excitation radiation reaching the detector, the optical system may be designed using filtering components, such as dichroic and band-pass filters, to provide discrimination between excitation and emission radiation wavelengths.
The process used to deposit a microarray of target and probe genetic material onto a substrate is conventionally referred to as spot placement. In the present state of the art, spot placement is performed by means of a gantry type computer-controlled robotic system. This conventional method typically requires additional procedures following the placement process to compensate for imprecise spot placement. For example, the operator may need to perform a low-resolution scan of the microarray sample to locate the spots of genetic material prior to performing the high-resolution scan used for quantitation.
Differential gene expression refers to a comparative experiment in which the gene expression of a known xe2x80x98controlxe2x80x99 sample is compared to the gene expression of a xe2x80x98testxe2x80x99 sample to determine the difference in gene expression levels. This comparison process determines the proportion of one gene identified with respect to another gene. Unique fluorophore materials are utilized (i.e., one fluorophore material for each gene) in gene expression experiments. Use of unique fluorophore materials makes it possible to view the information from each gene separately; otherwise no differential information could be obtained. This comparison process is not limited to the use of only one control sample and one test sample. In the present state of the art, five or more unique excitation wavelengths can be used to provide one control image and at least four additional test images.
A unique wavelength of light is used to excite a fluorescent emission from a corresponding fluorophore material. This produces an image file for the control sample and a separate image file for each test sample. In the image files, each spot is mapped to a corresponding brightness value as an indication of gene expression level. In way of example, the control image can be depicted using a green color palette, and the test image can be depicted in red. When the control and the test images are superimposed, certain superimposed spots appear yellow, with varying hue and brightness, in those locations where the corresponding control spot was green and the test spot was red. In the locations where only one of the control and test spots exhibited color, the superimposed spot would, accordingly, appear as an individual red or green spot. Alignment of the control and test images can be facilitated by computer software that provides a means to variably offset one image from the other by the use of appropriate keystrokes. Although this feature may allow the user to align the superimposed spots with more precision to provide a higher quality assessment, the process can still be tedious and lengthy.
Before an operator calculates the brightness of each hybridized spot and of the local background prior to quantitating the microarray sample, a mapping or pattern of the microarray spot locations is usually generated. The microarray mapping is a template used by the detection software to more efficiently search for the true locations of each spot in the pattern. For a relatively small number of spots (e.g., less than one hundred), the operator can usually locate each spot manually. For microarray patterns of more than one hundred spots, manual location becomes cumbersome, and for even larger arrays, the process of manual location becomes impractical. Moreover, the trend in the present state of the art is to develop automated methods of microarray inspection and it is desirable to provide efficiency and accuracy in the creation of the microarray configuration and in the methods of quantitation of the spots.
Several parameters can be used to fully describe a regular grid pattern: the numbers of rows and columns of spots, the distances between rows and columns, and the average diameter of each spot. The direct method of generating a regular grid pattern requires the manual entry of appropriate values for each of these parameters. This direct method, however, relies on having prior information as to the parameters of the microarray pattern and, because of the imprecision inherent in conventional spot placement methods (e.g., manual placement, robotic spot placement equipment) the parameter values will likewise be relatively imprecise, making the direct method even more difficult.
While the relevant art provides a method for performing the above procedures, there remains a need for improvements that offer advantages and capabilities not found in presently available methods of scanning, and it is a primary object of this invention to provide such improvements.
It is another object of the present invention to provide a method of scanning in which alignment is achieved quickly and accurately.
Other objects of the invention will be obvious, in part, and, in part, will become apparent when reading the detailed description to follow.
By using microarray substrates with one or more fiducial marks located at predetermined locations with respect to a microarray sample, the sample can be positioned and aligned with greater precision than with conventional systems in the performance of an alignment or quantitation procedure. The disclosed method uses the stored location(s) of the fiducial mark(s) to apply X- and Y-offsets, and rotations in the X-Y plane, to minimize the distance between all fiducial marks in all related images. This manipulation will have the effect of automatically registering features such as microarray spots that have been accurately placed relative to the fiducial mark locations.